The present invention features, inter alia, purified antibodies that selectively bind an isolated or recombinant histone deacetylase polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2, which comprises a histone deacetylase catalytic domain at amino acids 635 to 953 of SEQ ID NO: 2, purified antibodies that selectively bind a biologically active fragment of the polypeptide of SEQ ID NO: 2, which fragment exhibits histone deacetylase activity, transcription repression activity, and the ability to deacetylate cellular substrates, purified antibodies that selectively bind an isolated or recombinant histone deacetylase polypeptide encoded by a nucleotide sequence as set forth in SEQ ID NO: 1, purified antibodies that selectively bind an isolated or recombinant histone deacetylase polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 lacking a nuclear localization signal, and purified antibodies that selectively bind an isolated or recombinant histone deacetylase polypeptide having at least 95% amino acid sequence identity to SEQ ID NO: 2 and which exhibits histone deacetylase activity, transcription repression activity, and the ability to deacetylate cellular substrates.
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2. A purified antibody that selectively binds an isolated or recombinant histone deacetylase polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:2, wherein the antibody does not bind to HDRP consisting of the amino acid set forth in SEQ ID NO: 11.
1. A purified antibody that selectively binds an isolated or recombinant histone deacetylase polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, wherein the antibody does not bind to HDRP consisting of the amino acid set forth in SEQ ID NO: 11.
3. A purified antibody that selectively binds an isolated or recombinant histone deacetylase polypeptide, wherein said polypeptide is encoded by a nucleotide sequence as set forth in SEQ ID NO: 1, wherein the antibody does not bind to HDRP consisting of the amino acid set forth in SEQ ID NO: 11.
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This application is a divisional of U.S. application Ser. No. 11/202,268, filed on Aug. 10, 2005, now U.S. Pat. No. 7,244,604, which is a divisional of U.S. application Ser. No. 10/173,539, filed on Jun. 14, 2002, now U.S. Pat. No. 7,063,973, which claims the benefit of U.S. Provisional Application Ser. No. 60/298,173 filed on Jun. 14, 2001, U.S. Provisional Application No. 60/311,686 filed on Aug. 10, 2001 and U.S. Provisional Application No. 60/316,995, filed on Sep. 4, 2001. The entire teachings of the above applications are incorporated herein by reference in their entirety.
The invention was supported, in whole or in part, by grant CA-0974823 from the National Cancer Institute. The Government has certain rights in the invention.
The N-terminal tails of core histones are covalently modified by post-translational modifications, including acetylation and phosphorylation. Evidence suggests that these covalent modifications play important roles in several biological activities involving chromatin, e.g., transcription and replication. Histone deacetylases (HDACs) catalyze the removal of the acetyl group from the lysine residues in the N-terminal tails of nucleosomal core histones resulting in a more compact chromatin structure, a configuration that is generally associated with repression of transcription.
Five proteins and/or open reading frames in yeast (RPD3, HDA1, HOS1, HOS2 and HOS3) that share significant homology in the catalytic domain have been identified as HDACs based upon their sequence homology to human HDAC1. To date, eight HDACs have been identified in mammalian cells, and classified into two classes based on their structure and similarity to yeast RPD3 or HDA1 proteins. Recently, Sir2 family proteins that are structurally unrelated to the five proteins aforementioned have been identified as NAD-dependent HDACs. Class I HDACs are the yeast RPD3 homologs HDAC1, 2, 3, and 8, and are composed primarily of a catalytic domain. Class II HDACs are the yeast HDA1 homologs HDAC4, 5, 6, and 7. HDAC4, 5, and 7 contain a long non-catalytic N-terminal end and a C-terminal HDAC catalytic domain while HDAC6 has two HDAC catalytic domains.
It has also been determined that histone deacetylases can be sensitive to small molecules, including trichostatin A (TSA), trapoxin, and butyrate. For example, the yeast RPD3 and HDA1 and mammalian HDAC1, 2, 3, 4, 5, 6, 7 and 8 are sensitive to inhibition by trichostatin A (TSA). The Sir2 family HDACs, yeast HOS3 and Drosophila melanogaster dHDAC6, however, appear to be relatively insensitive to TSA. A class of hybrid bipolar compounds, such as suberoylanilide hydroxamic acid (SAHA) have also been shown to inhibit histone deacetylases and induce terminal differentiation and/or apoptosis in various transformed cells. Examples of such compounds can be found in U.S. Pat. No. 5,369,108, issued on Nov. 29, 1994, U.S. Pat. No. 5,700,811, issued on Dec. 23, 1997, and U.S. Pat. No. 5,773,474, issued on Jun. 30, 1998 to Breslow et al., as well as U.S. Pat. No. 5,055,608, issued on Oct. 8, 1991, and U.S. Pat. No. 5,175,191, issued on Dec. 29, 1992 to Marks et al., the entire content of all of which are hereby incorporated by reference.
The identification of the mechanisms by which histones are deacetylated, and the characterization of histone deacetylase function would be of great benefit in understanding how gene transcription is controlled, how the cell cycle is regulated, and how cells are signaled to undergo terminal differentiation and/or apoptosis. Elucidation of such mechanisms can lead to improved therapeutics for many diseases, in particular those characterized by cell proliferation or a lack of cell differentiation or apoptosis, for example, cancer.
The present invention relates to isolated or recombinant histone deacetylase polypeptides, and isolated histone deacetylase nucleic acid molecules encoding those polypeptides, as well as vectors and cells containing those isolated nucleic acid molecules.
In one aspect of the invention, the isolated or recombinant histone deacetylase polypeptide is selected from a) an isolated or recombinant polypeptide comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; and b) a polypeptide having at least 60% sequence identity with any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In one embodiment, the isolated or recombinant histone deacetylase polypeptide consists of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In another embodiment, the isolated or recombinant histone deacetylase polypeptide is mammalian; preferably, the isolated or recombinant histone deacetylase polypeptide is human.
In another aspect, the invention features an isolated nucleic acid molecule selected from a) an isolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; b) a complement of an isolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; c) an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; d) a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; e) a nucleic acid that is hybridizeable under high stringency conditions to a nucleic acid molecule that encodes any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a complement thereof; or f) a nucleic acid molecule that is hybridizeable under high stringency conditions to a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; and g) an isolated nucleic acid molecule that has at least 55% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a complement thereof. In one embodiment, the isolated nucleic acid molecule consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In another embodiment, the isolated nucleic acid molecule is mammalian; preferably, the isolated nucleic acid molecule is human.
In other aspects, the invention features a vector comprising the isolated histone deacetylase nucleic acid molecule described above, a cell comprising the vector, and a cell comprising the isolated histone deacetylase nucleic acid molecule described above.
In another aspect, the invention features a purified antibody that selectively binds a histone deacetylase polypeptide described above.
In yet another aspect, the invention features a method of identifying a compound that modulates expression of a histone deacetylase nucleic acid molecule described above. The method comprises the steps of a) contacting the nucleic acid molecule with a candidate compound under conditions suitable for expression; and b) assessing the level of expression of the nucleic acid molecule. A candidate compound that increases or decreases expression of the nucleic acid molecule relative to a control is a compound that modulates expression of the nucleic acid molecule. In one embodiment, the method is carried out in a cell or animal. In another embodiment, the method is carried out in a cell free system.
The invention also features a method of treating a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, for example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, and lung cancer and myeloproliferative disorders, including polycythemia vera, essential thrombocythemia, agnogenic myeloid metaplasia, and chronic myelogenous leukemia in an individual, comprising administering a compound identified by the above method.
In still another aspect, the invention features a method of identifying a compound that modulates the enzymatic activity of the histone deacetylase polypeptide described above. The method comprises the steps of a) contacting the polypeptide with a candidate compound under conditions suitable for enzymatic reaction; and b) assessing the activity level of the polypeptide. A candidate compound that increases or decreases the activity level of the polypeptide relative to a control is a compound that modulates the enzymatic activity of the polypeptide. In one embodiment, the method is carried out in a cell or animal. In another embodiment, the method is carried out in a cell free system.
In yet another embodiment, the polypeptide is further contacted with a substrate for the polypeptide, wherein the substrate is selected from the group consisting of a cell proliferation disease binding agent, an apoptotic disease binding agent, and a cell differentiation disease binding agent. In one embodiment, the candidate compound is an inhibitor. In another embodiment, candidate compound is an activator.
In another aspect, the invention features a method of identifying a compound that modulates the transcriptional repression activity of the histone deacetylase polypeptide described above. The method comprises the steps of a) contacting the polypeptide with a candidate compound under conditions suitable for a transcriptional repression reaction; and b) assessing the transcriptional repression activity level of the polypeptide. A candidate compound that increases or decreases the transcriptional repression activity level of the polypeptide relative to a control is a compound that modulates the transcriptional repression activity of the polypeptide. In one embodiment, the method is carried out in a cell or animal. In another embodiment, the method is carried out in a cell free system.
In yet another embodiment, the polypeptide is further contacted with a substrate for the polypeptide, wherein the substrate is selected from the group consisting of a cell proliferation disease binding agent, an apoptotic disease binding agent, and a cell differentiation disease binding agent. In one embodiment, the candidate compound is an inhibitor. In another embodiment, candidate compound is an activator.
In another aspect, the invention features a method of identifying a compound that modulates expression of a histone deacetylase nucleic acid molecule described above. The method comprises the steps of a) providing a nucleic acid molecule comprising a promoter region of the histone deacetylase nucleic acid molecule described above, or part of such a promoter region, operably linked to a reporter gene; b) contacting the nucleic acid molecule or with a candidate compound; and c) assessing the level of the reporter gene. A candidate compound that increases or decreases expression of the reporter gene relative to a control is a compound that modulates expression of the histone deacetylase nucleic acid molecule described above. In one embodiment, the method is carried out in a cell.
In still another aspect, the invention features a method of identifying a polypeptide that interacts with a histone deacetylase polypeptide described above in a yeast two-hybrid system. The method comprises the steps of a) providing a first nucleic acid vector comprising a nucleic acid molecule encoding a DNA binding domain and the histone deacetylase polypeptide described above; b) providing a second nucleic acid vector comprising a nucleic acid encoding a transcription activation domain and a nucleic acid encoding a test polypeptide; c) contacting the first nucleic acid vector with the second nucleic acid vector in a yeast two-hybrid system; and d) assessing transcriptional activation in the yeast two-hybrid system. An increase in transcriptional activation relative to a control indicates that the test polypeptide is a polypeptide that interacts with the histone deacetylase polypeptide described above.
The invention also features a pharmaceutical composition comprising a histone deacetylase polypeptide described above.
In addition, the present invention features a method of diagnosing a cell proliferation disease, an apoptotic disease, or a cell differentiation disease in a subject. The method comprises the steps of a) obtaining a sample from the subject; and b) assessing the level of activity or expression of the histone deacetylase polypeptide described above or the level of the nucleic acid molecule described above in the sample. If the level is increased relative to a control, then the subject has an increased likelihood of having a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, and if the level is decreased relative to a control, then the subject has a decreased likelihood of having a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. In one embodiment, the polypeptide level is assayed using immunohistochemistry techniques. In another embodiment, the nucleic acid molecule level is assayed using in situ hybridization techniques.
Compounds and/or polypeptides identified in the above-described screening methods are also part of the present invention.
A protein designated HDRP (See Zhou et al., Proc. Natl. Acad. Sci. USA, 97:1056-1061 (2000)) (also called MITR (See Sparrow et al., EMBO J. 18:5085-5098(1999); Zhang et al, J. Biol. Chem., 276:35-39 (2001); and Zhang et al., Proc. Natl. Acad. Sci. USA, 98:7354-7359 (2001)) that is 50% identical to the N-terminal domains of histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5) was recently identified. The cloning and characterization of a novel histone deacetylase, HDAC9, of which HDRP is an alternatively spliced isoform is described herein. The cDNA sequence of HDAC9 is shown in
Polypeptides of the Invention
The present invention features isolated or recombinant HDAC9 polypeptides, HDAC9a polypeptides, HDAC9(ΔNLS) polypeptides, HDAC9a(ΔNLS) polypeptides, and HDRP(ΔNLS) polypeptides, and fragments, derivatives, and variants thereof, as well as polypeptides encoded by nucleotide sequences described herein (e.g., other variants). As used herein, the term “polypeptide” refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides, and proteins are included within the definition of a polypeptide.
As used herein, a polypeptide is said to be “isolated,” “substantially pure,” or “substantially pure and isolated” when it is substantially free of cellular material, when it is isolated from recombinant or non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. Typically, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide is isolated, substantially pure, or substantially pure and isolated when it has a relative increased concentration or activity of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), in comparison to total HDAC concentration or activity. Preferably the increased activity or concentration of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is at least 2-fold, more preferably, at least 5-fold, and most preferably, at least 10 fold, in comparison to total HDAC concentration or activity. In addition, a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell (e.g.; in a “fusion protein”) and still be “isolated,” “substantially pure,” or “substantially pure and isolated.” An isolated, substantially pure, or substantially pure and isolated polypeptide may be obtained, for example, using affinity purification techniques described herein, as well as other techniques described herein and known to those skilled in the art.
By a “histone deacetylase polypeptide” is meant a polypeptide having histone deacetylase activity, transcription repression activity, and/or the ability to deacetylate other substrates, for example, transcription factors, including p53, CoRest, E2F, GATA-1, TFIIe, and TFIIF that normally have a nuclear or cytoplasmic location in a cell. A histone deacetylase polypeptide is also a polypeptide whose activity can be inhibited by molecules having HDAC inhibitory activity. These molecules fall into four general classes: 1) short-chain fatty acids (e.g., 4-phenylbutyrate and valproic acid); 2) hydroxamic acids (e.g. SAHA, Pyroxamide, trichostatin A (TSA), oxamflatin and CHAPs, such as, CHAP1 and CHAP 31); 3) cyclic tetrapeptides (Trapoxin A, Apicidin and Depsipeptide (FK-228, also known as FR9011228); 4) benzamides (e.g., MS-275); and other compounds such as Scriptaid. Examples of such compounds can be found in U.S. Pat. No. 5,369,108, issued on Nov. 29, 1994, U.S. Pat. No. 5,700,811, issued on Dec. 23, 1997, and U.S. Pat. No. 5,773,474, issued on Jun. 30, 1998 to Breslow et al., U.S. Pat. No. 5,055,608, issued on Oct. 8, 1991, and U.S. Pat. No. 5,175,191, issued on Dec. 29, 1992 to Marks et al., as well as, Yoshida et al., Bioessays 17, 423-430 (1995), Saito et al., PNAS USA 96, 4592-4597, (1999), Furamai et al., PNAS USA 98 (1), 87-92 (2001), Komatsu et al., Cancer Res. 61(11), 4459-4466 (2001), Su et al., Cancer Res. 60, 3137-3142 (2000), Lee et al., Cancer Res. 61(3), 931-934 and Suzuki et al. J. Med. Chem. 42(15), 3001-3003 (1999) the entire content of all of which are hereby incorporated by reference. Examples of such histone deacetylase polypeptides include HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), HDRP(ΔNLS); a substantially pure polypeptide comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; and a polypeptide having preferably at least 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, as determined using the BLAST program and parameters described herein.
In one embodiment, the histone deacetylase polypeptide has histone deacetylase activity, transcription repression activity, the ability to deacetylate substrates, or is inhibited by trichostatin A or a hybrid polar compound such as SAHA. In another embodiment, the HDAC9(ΔNLS) polypeptide has any two of the above biological activities. In still another embodiment, the HDAC9(ΔNLS) polypeptide has any three of the above biological activities. In yet another embodiment, the HDAC9(ΔNLS) polypeptide has all of the above biological activities.
An HDAC9 polypeptide is a histone deacetylase polypeptide as described above. An HDAC9 polypeptide preferably has at least 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% sequence identity to SEQ ID NO: 2, as determined using the BLAST program and parameters described herein. An HDAC9 polypeptide is also a polypeptide that comprises the amino acids encoded by exons 23, 24, 25 and/or 26, and that does not comprise the amino acids encoded by exon 13 of the HDAC9 nucleic acid sequence, as shown in
An HDAC9a polypeptide is a histone deacetylase polypeptide as described above. An HDAC9a polypeptide preferably has at least 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% sequence identity to SEQ ID NO: 4, as determined using the BLAST program and parameters described herein. An HDAC9a polypeptide is also a polypeptide that comprises the amino acids encoded by exon 22, and that does not comprise the amino acids encoded by exons 13, 23, 24, 25, or 26 of the HDAC9 nucleic acid sequence, as shown in
An HDAC9(ΔNLS) is a histone deacetylase polypeptide as described above. An HDAC9(ΔNLS) polypeptide does not comprise a nuclear localization signal (NLS). An HDAC9(ΔNLS) polypeptide preferably has at least 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% sequence identity to SEQ ID NO: 6, as determined using the BLAST program and parameters described herein. An HDAC9(ΔNLS) polypeptide is also a polypeptide that comprises the amino acids encoded by exons 23, 24, 25, and/or 26, and that does not comprise the amino acids encoded by exons 7 or 13 of the HDAC9 nucleic acid sequence, as shown in
An HDAC9a(ΔNLS) polypeptide is a histone deacetylase polypeptide as described above. An HDAC9a(ΔNLS) does not comprise a nuclear localization signal (NLS). An HDAC9a(ΔNLS) polypeptide preferably has at least 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% sequence identity to SEQ ID NO: 8; as determined using the BLAST program and parameters described herein. An HDAC9a(ΔNLS) polypeptide is also a polypeptide that comprises the amino acids encoded by exon 22, and that does not comprise the amino acids encoded by exons 7, 13, 23, 24, 25, or 26 of the HDAC9 nucleic acid sequence, as shown in
An HDRP(ΔNLS) polypeptide is a histone deacetylase polypeptide as described above. An HDRP(ΔNLS) does not comprise a nuclear localization signal (NLS). An HDRP(ΔNLS) polypeptide preferably has at least 60%, more preferably, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% sequence identity to SEQ ID NO: 10, as determined using the BLAST program and parameters described herein. An HDRP(ΔNLS) polypeptide is also a polypeptide that does not comprise the amino acids encoded by exons 7 or 13-26 of the HDAC9 nucleic acid sequence, as shown in
The polypeptides of the invention can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful. The critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity. In one embodiment, the language “substantially free of cellular material” includes preparations of the polypeptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation. The language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
In one embodiment, a polypeptide of the invention comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and complements and portions thereof, (e.g., a complement of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or a portion of any one of SEQ ID NO: 1 or SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9).
The polypeptides of the invention also encompass fragments and sequence variants. Variants include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other variants. Variants also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and complements and portions thereof, or having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of nucleotide sequences encoding any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. Variants also include polypeptides substantially homologous or identical to these polypeptides but derived from another organism, i.e., an ortholog. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by recombinant methods.
As used herein, two polypeptides (or a region of the polypeptides) are substantially homologous or identical when the amino acid sequences are at least about 60-65%, typically at least about 70-75%, more typically at least about 80-85%, and most typically greater than about 90-95% or more homologous or identical. A substantially identical or homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid molecule hybridizing to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a portion thereof, under stringent conditions as more particularly described herein, or will be encoded by a nucleic acid molecule hybridizing to a nucleic acid sequence encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or portion thereof, under stringent conditions as more particularly described herein.
The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100). In certain embodiments, the length of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) amino acid or nucleotide sequence aligned for comparison purposes is at least 30%, preferably, at least 40%, more preferably, at least 60%, and even more preferably, at least 70%, 80%, 90%, or 100% of the length of the reference sequence, for example, those sequences provided in
Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl. Biosci., 10: 3-5 (1994); and FASTA described in Pearson and Lipman, Proc. Natl. Acad. Sci USA, 85: 2444-8 (1988).
In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3 or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using a gap weight of 50 and a length weight of 3.
The invention also encompasses HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, and HDRP(ΔNLS) polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, or HDRP(ΔNLS) polypeptide encoded by a nucleic acid molecule of the invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247: 1306-1310 (1990).
A variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. Further, variant polypeptides can be fully functional or can lack function in one or more activities, for example, in histone deacetylase activity or transcription repression activity. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations or a substitution, insertion, inversion, or deletion in a critical residue or critical region, such critical regions include the HDAC domains, which provide the polypeptide with deacetylase activity, as shown in the nucleic acid sequences of
Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science, 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each of the residues in the molecule (one mutation per molecule). The resulting mutant molecules are then tested for biological activity in vitro. Sites that are critical for polypeptide activity can also be determined by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (See Smith et al., J. Mol. Biol., 224: 899-904 (1992); and de Vos et al. Science, 255: 306-312 (1992)).
The invention also includes HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) polypeptide fragments of the polypeptides of the invention. Fragments can be derived from a polypeptide comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or from a polypeptide encoded by a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 or a portion thereof and the complements thereof or other variants. The present invention also encompasses fragments of the variants of the polypeptides described herein. Useful fragments include those that retain one or more of the biological activities of the polypeptide as well as fragments that can be used as an immunogen to generate polypeptide-specific antibodies.
Biologically active fragments (peptides that are, for example, 6, 9, 12, 15, 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, or more amino acids in length) can comprise a domain, segment, or motif, for example, an HDAC domain, that has been identified by analysis of the polypeptide sequence using well-known methods, e.g., signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, acylation sites, glycosylation sites, or phosphorylation sites.
Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment.
The invention thus provides chimeric or fusion polypeptides. These comprise an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, or HDRP(ΔNLS) polypeptide of the invention operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide. “Operatively linked” indicates that the polypeptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide. In one embodiment, the fusion polypeptide does not affect the function of the polypeptide per se. For example, the fusion polypeptide can be a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-terminus of the GST sequences. Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example, β-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, and Ig fusions. Such fusion polypeptides, particularly poly-His fusions, can facilitate the purification of recombinant polypeptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence. Therefore, in another embodiment, the fusion polypeptide contains a heterologous signal sequence at its N-terminus.
EP-A 0464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). In drug discovery, for example, human proteins have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists. (See Bennett et al., Journal of Molecular Recognition, 8: 52-58 (1995) and Johanson et al., The Journal of Biological Chemistry, 270,16: 9459-9471 (1995)). Thus, this invention also encompasses soluble fusion polypeptides containing a polypeptide of the invention and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclass (IgG, IgM, IgA, IgE).
A chimeric or fusion polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al., “Current Protocols in Molecular Biology,” John Wiley & Sons, (1998), the entire teachings of which are incorporated by reference herein). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.
The substantially pure, isolated, or substantially pure and isolated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, or HDRP(ΔNLS) polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. In one embodiment, the polypeptide is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector introduced into a host cell, and the polypeptide expressed in the host cell. The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
In general, HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9aΔNLS, and HDRP(ΔNLS) polypeptides of the present invention can be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using art-recognized methods. The polypeptides of the present invention can be used to raise antibodies or to elicit an immune response. The polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the polypeptide or a molecule to which it binds (e.g., a receptor or a ligand) in biological fluids. The polypeptides can also be used as markers for cells or tissues in which the corresponding polypeptide is preferentially expressed, either constitutively, during tissue differentiation, or in a diseased state. The polypeptides can be used to isolate a corresponding binding agent, and to screen for peptide or small molecule antagonists or agonists of the binding interaction. The polypeptides of the present invention can also be used as therapeutic agents.
Nucleic Acid Molecules of the Invention
The present invention also features isolated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules.
By a “histone deacetylase nucleic acid molecule” is meant a nucleic acid molecule that encodes a histone deacetylase polypeptide. Such histone nucleic acids include, for example, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule described in detail herein; an isolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; a complement of an isolated nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; a nucleic acid that is hybridizeable under high stringency conditions to a nucleic acid molecule that encodes any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a complement thereof; a nucleic acid molecule that is hybridizeable under high stringency conditions to a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; and an isolated nucleic acid molecule that has at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a complement thereof.
An HDAC9 nucleic acid molecule is a nucleic acid molecule that encodes an HDAC9 polypeptide. In one embodiment, the HDAC9 nucleic acid molecule is selected from: a nucleic acid molecule that comprises the nucleic acid sequence of SEQ ID NO: 1; a complement of an isolated nucleic acid comprising SEQ ID NO: 1; an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2; a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 2; a nucleic acid that is hybridizeable under high stringency conditions to a nucleic acid molecule that encodes SEQ ID NO: 2; a nucleic acid molecule that is hybridizeable under high stringency conditions to a nucleic acid comprising SEQ ID NO: 1; and an isolated nucleic acid molecule that has preferably, at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequence identity with SEQ ID NO: 1, as determined using the BLAST program and parameters described herein. In another embodiment, the HDAC9 nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO: 1.
An HDAC9a nucleic acid molecule is a nucleic acid molecule that encodes an HDAC9a polypeptide. An HDAC9a nucleic acid molecule preferably has at least 55%, sequence identity to SEQ ID NO: 3, In one embodiment, the HDAC9a nucleic acid molecule is selected from: a nucleic acid molecule that comprises the nucleic acid sequence of SEQ ID NO: 3; a complement of an isolated nucleic acid comprising SEQ ID NO: 3; an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 4; a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 4; a nucleic acid that is hybridizeable under high stringency conditions to a nucleic acid molecule that encodes SEQ ID NO: 4; a nucleic acid molecule that is hybridizeable under high stringency conditions to a nucleic acid comprising SEQ ID NO: 3; and an isolated nucleic acid molecule that has preferably, at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequence identity with SEQ ID NO: 3 or a complement thereof, as determined using the BLAST program and parameters described herein. In another embodiment, the HDAC9a nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO: 3.
An HDAC9(ΔNLS) nucleic acid molecule is a nucleic acid molecule that encodes an HDAC9(ΔNLS) polypeptide. In one embodiment, the HDAC9(ΔNLS) nucleic acid molecule is selected from: a nucleic acid molecule that comprises the nucleic acid sequence of SEQ ID NO: 5; a complement of an isolated nucleic acid comprising SEQ ID NO: 5; an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 6; a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 6; a nucleic acid that is hybridizeable under high stringency conditions to a nucleic acid molecule that encodes SEQ ID NO: 6; a nucleic acid molecule that is hybridizeable under high stringency conditions to a nucleic acid comprising SEQ ID NO: 5; and an isolated nucleic acid molecule that has preferably, at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequence identity with SEQ ID NO: 5 or a complement thereof, as determined using the BLAST program and parameters described herein. In another embodiment, the HDAC9(ΔNLS) nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO: 5.
An HDAC9a(ΔNLS) nucleic acid molecule is a nucleic acid molecule that encodes an HDAC9a(ΔNLS) polypeptide. In one embodiment, the HDAC9a(ΔNLS) nucleic acid molecule is selected from: a nucleic acid molecule that comprises the nucleic acid sequence of SEQ ID NO: 7; a complement of an isolated nucleic acid comprising SEQ ID NO: 7; an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 8; a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 8; a nucleic acid that is hybridizeable under high stringency conditions to a nucleic acid molecule that encodes SEQ ID NO: 8; a nucleic acid molecule that is hybridizeable under high stringency conditions to a nucleic acid comprising SEQ ID NO: 7; and an isolated nucleic acid molecule that has preferably, at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequence identity with SEQ ID NO: 7 or a complement thereof, as determined using the BLAST program and parameters described herein. In another embodiment, the HDAC9a(ΔNLS) nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO: 7.
An “HDRP(ΔNLS) nucleic acid molecule” is a nucleic acid molecule that encodes an HDRP(ΔNLS) polypeptide. In one embodiment, the HDRP(ΔNLS) nucleic acid molecule is selected from: a nucleic acid molecule that comprises the nucleic acid sequence of SEQ ID NO: 9; a complement of an isolated nucleic acid comprising SEQ ID NO: 9; an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 10; a complement of an isolated nucleic acid encoding a histone deacetylase polypeptide of SEQ ID NO: 10; and an isolated nucleic acid molecule that has preferably, at least 55%, more preferably, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, and most preferably, 95% or 99% sequence identity with SEQ ID NO: 9 or a complement thereof, as determined using the BLAST program and parameters described herein. In another embodiment, the HDRP(ΔNLS) nucleic acid molecule consists of the nucleic acid sequence of SEQ ID NO: 9.
The isolated nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA. DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense, strand or the non-coding, or antisense, strand. The nucleic acid molecule can include all or a portion of the coding sequence of the gene and can further comprise additional non-coding sequences such as introns and non-coding 3′ and 5′ sequences (including regulatory sequences, for example). Additionally, the nucleic acid molecule can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide. Such sequences include, but are not limited to, those that encode a glutathione-S-transferase (GST) fusion protein and those that encode a hemagglutinin A (HA) polypeptide marker from influenza.
An “isolated,” “substantially pure,” or “substantially pure and isolated” nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA or cDNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system, or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example, as determined by agarose gel electrophoresis or column chromatography such as HPLC. Preferably, an isolated nucleic acid molecule comprises at least about 50, 80, or 90% (on a molar basis) of all macromolecular species present.
With regard to genomic DNA, the term “isolated” also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of “isolated” as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution. “Isolated” nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Therefore, recombinant DNA contained in a vector are included in the definition of “isolated” as used herein.
Isolated nucleotide molecules also include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution. In vivo and in vitro RNA transcripts of the DNA molecules of the present invention are also encompassed by “isolated” nucleotide sequences. Such isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis.
The present invention also pertains to variant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules that are not necessarily found in nature but that encode an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. Thus, for example, DNA molecules that comprise a sequence that is different from the naturally-occurring HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleotide sequence but which, due to the degeneracy of the genetic code, encode an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the present invention are also the subject of this invention.
The invention also encompasses HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleotide sequences encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. Such variants can be naturally-occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. Intended variations include, but are not limited to, addition, deletion, and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. In one preferred embodiment, the nucleotide sequences are fragments that comprise one or more polymorphic microsatellite markers.
Other alterations of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, and carbamates), charged linkages (e.g., phosphorothioates or phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine or psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequences via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
The invention also pertains to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules that specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide). In one embodiment, the invention includes variants described herein that hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and the complement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In another embodiment, the invention includes variants described herein that hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2 (HDAC9), SEQ ID NO: 4 (HDAC9a), SEQ ID NO: 6 (HDAC9(ΔNLS)), SEQ ID NO: 8 (HDAC9a(ΔNLS)), or SEQ ID NO: 10 (HDRP(ΔNLS)). In a preferred embodiment, the variant that hybridizes under high stringency hybridizations encodes a polypeptide that has a biological activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (e.g., histone deacetylase activity or transcription repression activity).
Such nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions). “Specific hybridization,” as used herein, refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed). “Stringency conditions” for hybridization is a term of art that refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, that permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity that is less than perfect (e.g., 70%, 75%, 85%, 95%). For example, certain high stringency conditions can be used that distinguish perfectly complementary nucleic acids from those of less complementarity. “High stringency conditions,” “moderate stringency conditions,” and “low stringency conditions” for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols in Molecular Biology (See Ausubel et al., supra, the entire teachings of which are incorporated by reference herein). The exact conditions that determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2×SSC or 0.1×SSC), temperature (e.g., room temperature, 42° C. or 68° C.), and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences, and the frequency of occurrence of subsets of that sequence within other non-identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules. Typically, conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions that will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined.
Exemplary conditions are described in Krause and Aaronson, Methods in Enzymology, 200:546-556 (1991). Also, in, Ausubel, et al., supra, which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each ° C. by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in Tm of 17° C. Using these guidelines, the washing temperature can be determined empirically for high, moderate, or low stringency, depending on the level of mismatch sought.
For example, a low stringency wash can comprise washing in a solution containing 0.2×SSC/0.1% SDS for 10 minutes at room temperature; a moderate stringency wash can comprise washing in a prewarmed solution (42° C.) solution containing 0.2×SSC/0.1% SDS for 15 minutes at 42° C.; and a high stringency wash can comprise washing in prewarmed (68° C.) solution containing 0.1×SSC/0.1% SDS for 15 minutes at 68° C. Furthermore, washes can be performed repeatedly or sequentially to obtain a desired result as known in the art. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
To determine the percent homology or identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide or nucleic acid molecule for optimal alignment with the other polypeptide or nucleic acid molecule). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared, as described above.
The present invention also provides isolated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and the complement of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. The nucleic acid fragments of the invention are at least about 15, preferably, at least about 18, 20, 23, or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, that encode antigenic polypeptides described herein are particularly useful, such as for the generation of antibodies as described above.
In a related aspect, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. “Probes” or “primers” are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. Such probes and primers include polypeptide nucleic acids, as described in Nielsen et al., Science, 254, 1497-1500 (1991). As also used herein, the term “primer” in particular refers to a single-stranded oligonucleotide that acts as a point of initiation of template-directed DNA synthesis using well-known methods (e.g., PCR, LCR) including, but not limited to those described herein.
Typically, a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and more typically about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous nucleotide sequence selected from: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, the complement of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and a sequence encoding an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
In preferred embodiments, a probe or primer comprises 100 or fewer nucleotides, preferably, from 6 to 50 nucleotides, and more preferably, from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence, preferably, at least 80% identical, more preferably, at least 90% identical, even more preferably, at least 95% identical, or even capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
The nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided in SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and/or SEQ ID NO: 10. For example, nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the nucleic acid sequences provided above and/or the complement of those sequences. Or such nucleic acid molecules may be designed based on nucleotide sequences encoding one or more of the amino acid sequences provided in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., (1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis et al., Academic Press, San Diego, Calif., (1990); Mattila et al., Nucleic Acids Res., 19: 4967 (1991); Eckert et al., PCR Methods and Applications, 1: 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford)); and U.S. Pat. No. 4,683,202. The nucleic acid molecules can be amplified using cDNA, mRNA, or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.
Other suitable amplification methods include the ligase chain reaction (LCR) (See Wu and Wallace, Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989)), and self-sustained sequence replication (See Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, that produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
The amplified DNA can be radiolabeled and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZIPLOX, or other suitable vector. Corresponding clones can be isolated, DNA can be obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art-recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. For example, the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York (1989)); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, (1988)). Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced, and further characterized.
Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and/or the complement of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and/or a portion of those sequences, and/or the complement of those portion or sequences, and/or a sequence encoding the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or encoding a portion of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. Such antisense nucleic acid molecules can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).
In general, the isolated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid sequences of the invention can be used as molecular weight markers on Southern blots, and as chromosome markers that are labeled to map related gene positions. The nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify genetic disorders (e.g., a predisposition for or susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease), and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample. The nucleic acid molecules of the present invention can also be used as therapeutic agents.
By a “cell proliferation disease” is meant a disease that is caused by or results in undesirably high levels of cell division, undesirably low levels of apoptosis, or both. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, and lung cancer are all examples of cell proliferation diseases. Myeloproliferative disorders, including polycythemia vera, essential thrombocythemia, agnogenic myeloid metaplasia, and chronic myelogenous leukemia are also cell proliferation diseases.
By a “cell differentiation disease” is meant a disease that is caused by or results in undesirably low levels of cell differentiation, or by undesirably high levels of cell differentiation. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, and lung cancer are all examples of cell differentiation diseases. Myeloproliferative disorders, including polycythemia vera, essential thrombocythemia, agnogenic myeloid metaplasia, and chronic myelogenous leukemia are also cell differentiation diseases.
By an “apoptotic disease” is meant a condition in which the apoptotic response is abnormal. This may pertain to a cell or a population of cells that does not undergo cell death under appropriate conditions. For example, normally a cell will die upon exposure to apoptotic-triggering agents, such as chemotherapeutic agents, or ionizing radiation. When, however, a subject has an apoptotic disease, for example, cancer, the cell or a population of cells may not undergo cell death in response to contact with apoptotic-triggering agents. In addition, a subject may have an apoptotic disease when the occurrence of cell death is too low, for example, when the number of proliferating cells exceeds the number of cells undergoing cell death, as occurs in cancer when such cells do not properly differentiate.
An apoptotic disease may also be a condition characterized by the occurrence of undesirably high levels of apoptosis. For example, certain neurodegenerative diseases, including but not limited to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, restenosis, stroke, and ischemic brain injury are apoptotic diseases in which neuronal cells undergo undesired cell death.
Other diseases for which the polypeptides and nucleic acid molecules of the present invention may be useful for diagnosing and/or treating include, but are not limited to Huntington's disease.
The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid molecules of the present invention can further be used to derive primers for genetic fingerprinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses. Portions or fragments of the nucleotide sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.
In addition, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization, or therapeutic use, or as markers for tissues in which the corresponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states. The nucleic acid sequences can additionally be used as reagents in the screening and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.
Standard techniques, such as the polymerase chain reaction (PCR) and DNA hybridization, may be used to clone HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) homologs in other species, for example, mammalian homologs. HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) homologs may be readily identified using low-stringency DNA hybridization or low-stringency PCR with human HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) probes or primers. Degenerate primers encoding human HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides may be used to clone HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) homologs by RT-PCR.
Alternatively, additional HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) homologs can be identified by utilizing consensus sequence information for HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides to search for similar polypeptides in other species. For example, polypeptide databases for other species can be searched for proteins with the HDAC domains described herein. Candidate polypeptides containing such a motif can then be tested for their HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activities, using methods described herein.
Expression of the Nucleic Acid Molecules of the Invention
Another aspect of the invention pertains to nucleic acid constructs containing an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule, for example, one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and the complement of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 (or portions thereof). Yet another aspect of the invention pertains to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) nucleic acid constructs containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. The constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted in a sense or antisense orientation.
As used herein, the term “vector” or “construct” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
Preferred recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of polypeptide desired. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides, encoded by nucleic acid molecules as described herein.
The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells, such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast, or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, or methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule of the invention has been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered. Such animals are useful for studying the function and/or activity of the nucleotide sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity.
As used herein, a “transgenic animal” is a non-human animal, preferably, a mammal, more preferably, a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably, a mammal, more preferably, a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191, and in Hogan, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986)). Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, Current Opinion in Bio/Technology, 2:823-829 (1991) and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al., Nature, 385:810-813 (1997) and PCT Publication Nos. WO 97/07668 and WO 97/07669.
Antibodies of the Invention
Polyclonal and/or monoclonal antibodies that selectively bind one form of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide but not another form of the polypeptide are also provided. Antibodies are also provided that bind a portion of either the variant or reference HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide that contains the polymorphic site or sites.
In another aspect, the invention provides antibodies to each of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) polypeptides and polypeptide fragments of the invention, e.g., having an amino acid sequence encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or a portion thereof, or having an amino acid sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or another variant, or portion thereof).
The term “purified antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that selectively binds an antigen. A molecule that selectively binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample that naturally contains the polypeptide. Preferably the antibody is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it naturally associated. More preferably, the antibody preparation is at least 75% or 90%, and most preferably, 99%, by weight, antibody. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments that can be generated by treating the antibody with an enzyme such as pepsin.
The invention provides polyclonal and monoclonal antibodies that selectively bind to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention. The term “monoclonal antibody” or “monoclonal antibody composition,” as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention or fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature, 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today, 4:72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, Coligan et al., (eds.) John Wiley & Sons, Inc., New York, N.Y. (1994)). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody to a polypeptide of the invention (see, e.g., Current Protocols in Immunology, supra; Galfre et al., (1977) Nature, 266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); and Lerner, Yale J. Biol. Med., 54:387-402 (1981)). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods that also would be useful.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology, 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); and Griffiths et al., EMBO J., 12:725-734 (1993).
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
In general, antibodies of the invention (e.g., a monoclonal antibody) can be used to isolate an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation. A polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells. Moreover, an antibody specific for an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
The antibodies of the present invention can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S, and 3H.
Diagnostic and Screening Assays of the Invention
The present invention also pertains to diagnostic assays for assessing HDAC 9 HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) gene expression, or for assessing activity of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides of the invention. In one embodiment, the assays are used in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, or is at risk for (has a predisposition for or a susceptibility to) developing a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. The invention also provides for prognostic (or predictive) assays for determining whether an individual is susceptible to developing a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. For example, mutations in the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of symptoms associated with a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
Another aspect of the invention pertains to assays for monitoring the influence of agents, or candidate compounds (e.g., drugs or other agents) on the nucleic acid molecule expression or biological activity of polypeptides of the invention, as well as to assays for identifying candidate compounds that bind to an HDAC9, HDAC9a polypeptide, an HDAC9(ΔNLS) polypeptide, an HDAC9a(ΔNLS) polypeptide, or an HDRP(ΔNLS) polypeptide. These and other assays and agents are described in further detail in the following sections.
Diagnostic Assays
HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecules, probes, primers, polypeptides, and antibodies to an HDAC9, an HDAC9a protein, an HDAC9(ΔNLS) protein, an HDAC9a(ΔNLS) protein, or an HDRP(ΔNLS) protein can be used in methods of diagnosis of a susceptibility to, or likelihood of having a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, as well as in kits useful for diagnosis of a susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
In one embodiment of the invention, diagnosis of a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease is made by detecting a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). The polymorphism can be a mutation in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), such as the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift mutation; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of the gene; duplication of all or a part of the gene; transposition of all or a part of the gene; or rearrangement of all or a part of the gene, or a change in the expression pattern of the various HDAC9 isoforms. More than one such mutation may be present in a single nucleic acid molecule.
Such sequence changes cause a mutation in the polypeptide encoded by HDAC9, HDAC9a, HDAC9(NLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). For example, if the mutation is a frame shift mutation, the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease can be a synonymous mutation in one or more nucleotides (i.e., a mutation that does not result in a change in the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide). Such a polymorphism may alter sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the nucleic acid molecule. HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) that has any of the mutations described above is referred to herein as a “mutant nucleic acid molecule.”
In a first method of diagnosing a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, hybridization methods, such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see Ausubel, et al., supra). For example, a biological sample from a test subject (a “test sample”) of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for, or carrying a defect for, a cell proliferation disease, an apoptotic disease, or a cell differentiation disease (the “test individual”). The individual can be an adult, child, or fetus. The test sample can be from any source that contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract, or other organs. A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA sample is then examined to determine whether a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is present, and/or to determine which variant(s) encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is present. The presence of the polymorphism or variant(s) can be indicated by hybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A “nucleic acid probe,” as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or contains a nucleic acid encoding a particular variant of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). The probe can be any of the nucleic acid molecules described above (e.g., the entire nucleic acid molecule, a fragment, a vector comprising the gene, a probe, or primer, etc.).
To diagnose a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, a hybridization sample is formed by contacting the test sample containing HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), with at least one nucleic acid probe. A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can be all or a portion of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or the complement of SEQ ID NO: 1 or SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9; or can be a nucleic acid molecule encoding all or a portion of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. Other suitable probes for use in the diagnostic assays of the invention are described above (see. e.g., probes and primers discussed under the heading, “Nucleic Acids of the Invention”).
The hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). “Specific hybridization,” as used herein, indicates exact hybridization (e.g., with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly preferred embodiment, the hybridization conditions for specific hybridization are high stringency.
Specific hybridization, if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in the test sample, then HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) has the polymorphism, or is the variant, that is present in the nucleic acid probe. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or of the presence of a particular variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and is therefore diagnostic for a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
In Northern analysis (see Current Protocols in Molecular Biology, Ausubel, et al., supra), the hybridization methods described above are used to identify the presence of a polymorphism or of a particular variant, associated with a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. For Northern analysis, a test sample of RNA is obtained from the individual by appropriate means. Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or of the presence of a particular variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and is therefore diagnostic for a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
For representative examples of use of nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330.
Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described above. PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T, or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen et al., Bioconjugate Chemistry, 5 (1994), American Chemical Society, p. 1 (1994)). The PNA probe can be designed to specifically hybridize to a gene having a polymorphism associated with a susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. Hybridization of the PNA probe to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is diagnostic for a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
In another method of the invention, mutation analysis by restriction digestion can be used to detect a mutant nucleic acid molecule, or nucleic acid molecules containing a polymorphism(s), if the mutation or polymorphism in the gene results in the creation or elimination of a restriction site. A test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (and, if necessary, the flanking sequences) in the test sample of genomic DNA from the test individual. RFLP analysis is conducted as described (see Current Protocols in Molecular Biology, supra). The digestion pattern of the relevant DNA fragment indicates the presence or absence of the mutation or polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and therefore indicates the presence or absence of this decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
Sequence analysis can also be used to detect specific polymorphisms in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). A test sample of DNA or RNA is obtained from the test individual. PCR or other appropriate methods can be used to amplify the nucleic acid molecule, and/or its flanking sequences, if desired. The sequence of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or HDRP(ΔNLS), or a fragment of the any of those nucleic acid molecules, or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) cDNA, or a fragment of any of those cDNAs, or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA, or a fragment of any of those mRNAs, is determined, using standard methods. The sequence of the above gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the nucleic acid molecule, cDNA (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a nucleic acid sequence encoding the protein of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or a fragment thereof) or mRNA, as appropriate. The presence of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) indicates that the individual has a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
Allele-specific oligonucleotides can also be used to detect the presence of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki et al., Nature (London) 324:163-166 (1986)). An “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and that contains a polymorphism associated with a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. An allele-specific oligonucleotide probe that is specific for particular polymorphisms in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) can be prepared, using standard methods (see Current Protocols in Molecular Biology, supra).
To identify polymorphisms in the gene that are associated with a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease a test sample of DNA is obtained from the individual. PCR can be used to amplify all or a fragment of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and its flanking sequences. The DNA containing the amplified HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (or a fragment of any of those genes) is dot-blotted, using standard methods (see Current Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is then detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymorphism in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), and is therefore indicative of a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, can be used to identify polymorphisms in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). For example, in one embodiment, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also described as “GENECHIPS,” have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science, 251:767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854; PCT Publication No. WO 90/15070; Fodor et al., PCT Publication No. WO 92/10092, and U.S. Pat. No. 5,424,186, the entire teachings of each of which are incorporated by reference herein. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, the entire teachings of which are incorporated by reference herein.
Once an oligonucleotide array is prepared, a nucleic acid of interest is hybridized to the array and scanned for polymorphisms. Hybridization and scanning are generally carried out by methods described herein and also in, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein. In brief, a target nucleic acid sequence that includes one or more previously identified polymorphic markers is amplified by well known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the two strands of the target sequence both upstream and downstream from the polymorphism. Asymmetric PCR techniques may also be used. Amplified target, generally incorporating a label, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.
Although primarily described in terms of a single detection block, e.g., for detection of a single polymorphism, arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymorphisms. In alternate arrangements, it will generally be understood that detection blocks may be grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the array. For example, it may often be desirable to provide for the detection of those polymorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.
Additional descriptions of the use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and 5,837,832, the entire teachings of which are incorporated by reference herein.
Other methods of nucleic acid analysis can be used to detect polymorphisms in HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or variants encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). Representative methods include direct manual sequencing (Church and Gilbert Proc. Natl. Acad. Sci. USA 81: 1991-1995, (1988); Sanger et al., Proc. Natl. Acad. Sci. 74: 5463-5467 (1977); Beavis et al., U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad. Sci. USA 86: 232-236 (1991)), mobility shift analysis (Orita et al., Proc. Natl. Acad. Sci. USA 86: 2766-2770 (1989)), restriction enzyme analysis (Flavell et al., Cell 15: 25 (1978); Geever, et al., Proc. Natl. Acad. Sci. USA 78: 5081 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985)); RNase protection assays (Myers et al., Science 230: 1242 (1985)); use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele-specific PCR.
In another embodiment of the invention, diagnosis of a susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease can also be made by examining the level of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid, for example, using in situ hybridization techniques known to one skilled in the art, or by examining the level of expression, activity, and/or composition of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, by a variety of methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, immunohistochemistry, and immunofluorescence. A test sample from an individual is assessed for the presence of an alteration in the level of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid or in the expression and/or an alteration in composition of the polypeptide encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or for the presence of a particular variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). An alteration in expression of a polypeptide encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or an alteration in the qualitative polypeptide expression (e.g., expression of a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or variant thereof). In a preferred embodiment, diagnosis of a susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease is made by detecting a particular variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or a particular pattern of variants. Preferably, increased levels of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or increased expression or activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, relative to a control sample, for example, a sample known not to be associated with a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, indicates an increased susceptibility or likelihood that the individual has a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. Alternatively, decreased levels of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or decreased expression or activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, relative to a control sample, for example, a sample known not to be associated with a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, indicates a decreased susceptibility or likelihood that the individual has a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
Both quantitative and qualitative alterations can also be present. An “alteration” or “modulation” in the polypeptide expression, activity, or composition, as used herein, refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a control sample. A control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, is indicative of a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. Similarly, the presence of one or more different variants in the test sample, or the presence of significantly different amounts of different variants in the test sample, as compared with the control sample, is indicative of a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
It is understood that alterations or modulations in polypeptide expression or function can occur in varying degrees. For example, an alteration or modulation in expression can be an increase, for example, by at least 1.5-fold to 2-fold, at least 3-fold, or, at least 5-fold, relative to the control. Alternatively, the alteration or modulation in polypeptide expression can be a decrease, for example, by at least 10%, at least 40%, 50%, or 75%, or by at least 90%, relative to the control.
Various means of examining expression or composition of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide can be used, including spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al., U.S. Pat. No. 4,376,110) such as immunoblotting (see also Ausubel et al., supra; particularly chapter 10). For example, in one embodiment, an antibody capable of binding to the polypeptide (e.g., as described above), preferably an antibody with a detectable label, can be used. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled,” with regard to the antibody, is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reacting it with another reagent that is directly labeled. An example of indirect labeling is detection of a primary antibody using a fluorescently labeled secondary antibody.
Western blotting analysis, using an antibody as described above that specifically binds to a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an antibody that specifically binds to a non-mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an antibody that specifically binds to a particular variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), can be used to identify the presence in a test sample of a particular variant of a polypeptide encoded by a polymorphic or mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or the absence in a test sample of a particular variant or of a polypeptide encoded by a non-polymorphic or non-mutant gene. The presence of a polypeptide encoded by a polymorphic or mutant gene, or the absence of a polypeptide encoded by a non-polymorphic or non-mutant gene, is diagnostic for a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, as is the presence (or absence) of particular variants encoded by the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(NLS), or HDRP(ΔNLS) nucleic acid molecule.
In one embodiment of this method, the level or amount of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a test sample is compared with the level or amount of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, and is diagnostic for a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
Alternatively, the composition of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a test sample is compared with the composition of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in a control sample. A difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample (e.g., the presence of different variants), is diagnostic for a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. In another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample. A difference in the amount or level of the polypeptide in the test sample, compared to the control sample; a difference in composition in the test sample, compared to the control sample; or both a difference in the amount or level, and a difference in the composition, is indicative of a decreased susceptibility to a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
Kits (e.g., reagent kits) useful in the methods of diagnosis comprise components useful in any of the methods described herein, including, for example, hybridization probes or primers as described herein (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to a mutant or to non-mutant (native) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, means for amplification of nucleic acids comprising HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or means for analyzing the nucleic acid sequence of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or for analyzing the amino acid sequence of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, etc.
Screening Assays and Agents Identified Thereby
The invention provides methods (also referred to herein as “screening assays”) for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the invention, as well as for identifying the presence of a polypeptide encoded by a nucleic acid of the invention. In one embodiment, the presence (or absence) of a nucleic acid molecule of interest (e.g., a nucleic acid that has significant homology with a nucleic acid of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)) in a sample can be assessed by contacting the sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a nucleic acid having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, which may optionally comprise at least one polymorphism, or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or a fragment or variant of such nucleic acids), under stringent conditions as described above, and then assessing the sample for the presence (or absence) of hybridization. In a preferred embodiment, high stringency conditions are conditions appropriate for selective hybridization. In another embodiment, a sample containing the nucleic acid molecule of interest is contacted with a nucleic acid containing a contiguous nucleotide sequence (e.g., a primer or a probe as described above) that is at least partially complementary to a part of the nucleic acid molecule of interest (e.g., an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid), and the contacted sample is assessed for the presence or absence of hybridization. In a preferred embodiment, the nucleic acid containing a contiguous nucleotide sequence is completely complementary to a part of the nucleic acid molecule of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS).
In any of the above embodiments, all or a portion of the nucleic acid of interest can be subjected to amplification prior to performing the hybridization.
In another embodiment, the presence (or absence) of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, such as a polypeptide of the invention or a fragment or variant thereof, in a sample can be assessed by contacting the sample with an antibody that specifically binds to the polypeptide of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (e.g., an antibody such as those described above), and then assessing the sample for the presence (or absence) of binding of the antibody to the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide.
In another embodiment, the invention provides methods for identifying agents or compounds (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) that alter or modulate (e.g., increase or decrease) the activity of the polypeptides described herein, or that otherwise interact with the polypeptides herein. For example, such compounds can be compounds or agents that bind to polypeptides described herein (e.g., HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrates or agents); that have a stimulatory or inhibitory effect on, for example, activity of polypeptides of the invention; or that change (e.g., enhance or inhibit) the ability of the polypeptides of the invention to interact with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) binding agents; or that alter post-translational processing of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (e.g., agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface; or agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.). In one example, the binding agent is a cell proliferation disease binding agent, an apoptotic disease binding agent, or a cell differentiation disease binding agent. As used herein, by a “cell proliferation disease binding agent,” an “apoptotic disease binding agent,” or a “cell differentiation disease binding agent” is meant an agent as described herein that binds to a polypeptide of the present invention and modulates a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. The modulation can be an increase or a decrease in the severity or progression of the disease. In addition, a cell proliferation disease binding agent, an apoptotic disease binding agent, or a cell differentiation disease binding agent includes an agent that binds to a polypeptide that is upstream (earlier) or downstream (later) of the cell signaling events mediated by a polypeptide of the present invention, and thereby modulates the overall activity of the signaling pathway; in turn, the disease state is modulated.
The candidate compound can cause an increase in the activity of the polypeptide. For example, the activity of the polypeptide can be increased by at least 1.5-fold to 2-fold, at least 3-fold, or, at least 5-fold, relative to the control. Alternatively, the polypeptide activity can be a decrease, for example, by at least 10%, at least 20%, 40%, 50%, or 75%, or by at least 90%, relative to the control.
In one embodiment, the invention provides assays for screening candidate compounds or test agents to identify compounds that bind to or modulate the activity of polypeptides described herein (or biologically active portion(s) thereof), as well as agents identifiable by the assays. As used herein, a “candidate compound” or “test agent” is a chemical molecule, be it naturally-occurring or artificially-derived, and includes, for example, peptides, proteins, synthesized molecules, for example, synthetic organic molecules, naturally-occurring molecule, for example, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
In general, candidate compounds for uses in the present invention may be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, e.g., from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are generated, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. For example, candidate compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des., 12: 145 (1997)). Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activities should be employed whenever possible.
When a crude extract is found to modulate (i.e., stimulate or inhibit) the expression and/or activity of the nucleic acids and or polypeptides of the present invention, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having an activity that stimulates or inhibits nucleic acid expression, polypeptide expression, or polypeptide biological activity. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using animal models for diseases in which it is desirable to alter the activity or expression of the nucleic acids or polypeptides of the present invention.
In one embodiment, to identify candidate compounds that alter the biological activity, for example, the enzymatic activity or transcriptional repression activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, a cell, tissue, cell lysate, tissue lysate, or solution containing or expressing an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SE ID NO: 8, SEQ ID NO: 10, or another variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)), or a fragment or derivative thereof (as described above), can be contacted with a candidate compound to be tested under conditions suitable for enzymatic reaction or transcriptional repression reaction, as described herein.
Alternatively, the polypeptide can be contacted directly with the candidate compound to be tested. The level (amount) of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activity is assessed (e.g., the level (amount) of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activity is measured, either directly or indirectly), and is compared with the level of biological activity in a control (i.e., the level of activity of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or active fragment or derivative thereof in the absence of the candidate compound to be tested, or in the presence of the candidate compound vehicle only). If the level of the biological activity in the presence of the candidate compound differs, by an amount that is statistically significant, from the level of the biological activity in the absence of the candidate compound, or in the presence of the candidate compound vehicle only, then the candidate compound is a compound that alters the biological activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. For example, an increase in the level of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) enzymatic or transcriptional repression activity relative to a control, indicates that the candidate compound is a compound that enhances (is an agonist of) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Similarly, a decrease in the enzymatic level or transcriptional repression level of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity relative to a control, indicates that the candidate compound is a compound that inhibits (is an antagonist of) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. In another embodiment, the level of biological activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or derivative or fragment thereof in the presence of the candidate compound to be tested, is compared with a control level that has previously been established. A level of the biological activity in the presence of the candidate compound that differs from the control level by an amount that is statistically significant indicates that the compound alters HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) biological activity.
The present invention also relates to an assay for identifying compounds that alter the expression of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) that alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the nucleic acid molecule or that otherwise interact with the nucleic acids described herein, as well as compounds identifiable by the assays. For example, a solution containing a nucleic acid encoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide can be contacted with a candidate compound to be tested. The solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution that comprises elements necessary for transcription/translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired. The level and/or pattern of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression (e.g., the level and/or pattern of mRNA or of protein expressed, such as the level and/or pattern of different variants) is assessed, and is compared with the level and/or pattern of expression in a control (i.e., the level and/or pattern of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression in the absence of the candidate compound, or in the presence of the candidate compound vehicle only). If the level and/or pattern in the presence of the candidate compound differs, by an amount or in a manner that is statistically significant, from the level and/or pattern in the absence of the candidate compound, or in the presence of the candidate compound vehicle only, then the candidate compound is a compound that alters the expression of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). Enhancement of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression indicates that the candidate compound is an agonist of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Similarly, inhibition of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression indicates that the candidate compound is an antagonist of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. In another embodiment, the level and/or pattern of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide(s) (e.g., different variants) in the presence of the candidate compound to be tested, is compared with a control level and/or pattern that has previously been established. A level and/or pattern in the presence of the candidate compound that differs from the control level and/or pattern by an amount or in a manner that is statistically significant indicates that the candidate compound alters HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression.
In another embodiment of the invention, compounds that alter the expression of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or that otherwise interact with the nucleic acids described herein, can be identified using a cell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) gene operably linked to a reporter gene. After contact with a candidate compound to be tested, the level of expression of the reporter gene (e.g., the level of mRNA or of protein expressed) is assessed, and is compared with the level of expression in a control (i.e., the level of the expression of the reporter gene in the absence of the candidate compound, or in the presence of the candidate compound vehicle only). If the level in the presence of the candidate compound differs, by an amount or in a manner that is statistically significant, from the level in the absence of the candidate compound, or in the presence of the candidate compound vehicle only, then the candidate compound is a compound that alters the expression of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), as indicated by its ability to alter expression of a gene that is operably linked to the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) gene promoter. Enhancement of the expression of the reporter indicates that the compound is an agonist of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. Similarly, inhibition of the expression of the reporter indicates that the compound is an antagonist of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) activity. In another embodiment, the level of expression of the reporter in the presence of the candidate compound to be tested, is compared with a control level that has previously been established. A level in the presence of the candidate compound that differs from the control level by an amount or in a manner that is statistically significant indicates that the candidate compound alters HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression.
Compounds that alter the amounts of different variants encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (e.g., a compound that enhances activity of a first variant, and that inhibits activity of a second variant), as well as compounds that are agonists of activity of a first variant and antagonists of activity of a second variant, can easily be identified using these methods described above.
In other embodiments of the invention, assays can be used to assess the impact of a candidate compound on the activity of a polypeptide in relation to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate, for example, an inhibitor of histone deacetylase activity. These inhibitors fall into four general classes: 1) short-chain fatty acids (e.g., 4-phenylbutyrate and valproic acid); 2) hydroxamic acids (e.g., SAHA, Pyroxamide, trichostatin A (TSA), oxamflatin and CHAPs, such as, CHAP1 and CHAP 31); 3) cyclic tetrapeptides (Trapoxin A, Apicidin and Depsipeptide (FK-228, also known as FR9011228); 4) benzamides (e.g., MS-275); and other compounds such as Scriptaid. Examples of such assays and compounds can be found in U.S. Pat. No. 5,369,108, issued on Nov. 29, 1994, U.S. Pat. No. 5,700,811, issued on Dec. 23, 1997, and U.S. Pat. No. 5,773,474, issued on Jun. 30, 1998 to Breslow et al., U.S. Pat. No. 5,055,608, issued on Oct. 8, 1991, and U.S. Pat. No. 5,175,191, issued on Dec. 29, 1992 to Marks et al., as well as, Yoshida et al., supra; Saito et al., supra; Furamai et al., supra; Komatsu et al., supra; Su et al., supra; Lee et al., supra and Suzuki et al. supra, the entire content of all of which are hereby incorporated by reference.
In one example, a cell or tissue that expresses or contains a compound that interacts with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (herein referred to as an “HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate,” which can be a polypeptide or other molecule that interacts with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)) is contacted with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in the presence of a candidate compound, and the ability of the candidate compound to alter the interaction between HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) and the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP (ΔNLS) substrate is determined, for example, by assaying activity of the polypeptide. Alternatively, a cell lysate or a solution containing the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate, can be used. A compound that binds to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate can alter the interaction by interfering with, or enhancing the ability of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) to bind to, associate with, or otherwise interact with the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate.
Determining the ability of the candidate compound to bind to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate can be accomplished, for example, by coupling the candidate compound with a radioisotope or enzymatic label such that binding of the candidate compound to the polypeptide can be determined by detecting the labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, candidate compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
It is also within the scope of this invention to determine the ability of a candidate compound to interact with the polypeptide without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a candidate compound with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate without the labeling of either the candidate compound, HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate (McConnell et al., (1992) Science, 257: 1906-1912). As used herein, a “microphysiometer” (e.g., CYTOSENSOR) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between ligand and polypeptide.
In another embodiment of the invention, assays can be used to identify polypeptides that interact with one or more HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides, as described herein. For example, a yeast two-hybrid system such as that described by Fields and Song (Fields and Song, Nature 340: 245-246 (1989)) can be used to identify polypeptides that interact with one or more HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides. In such a yeast two-hybrid system, vectors are constructed based on the flexibility of a transcription factor that has two functional domains (a DNA binding domain and a transcription activation domain). If the two domains are separated but fused to two different proteins that interact with one another, transcriptional activation can be achieved, and transcription of specific markers (e.g., nutritional markers such as His and Ade, or color markers such as lacZ) can be used to identify the presence of interaction and transcriptional activation. For example, in the methods of the invention, a first vector is used that includes a nucleic acid encoding a DNA binding domain and an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, variant, or fragment or derivative thereof, and a second vector is used that includes a nucleic acid encoding a transcription activation domain and a nucleic acid encoding a polypeptide that potentially may interact with the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, variant, or fragment or derivative thereof (e.g., an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide substrate or receptor). Incubation of yeast containing the first vector and the second vector under appropriate conditions (e.g., mating conditions such as used in the MATCHMAKER™ system from Clontech) allows identification of colonies that express the markers of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). These colonies can be examined to identify the polypeptide(s) that interact with the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or fragment or derivative thereof. Such polypeptides may be useful as compounds that alter the activity or expression of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, as described above.
In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate, or other components of the assay on a solid support, in order to facilitate separation of complexed from uncomplexed forms of one or both of the polypeptides, as well as to accommodate automation of the assay. Binding of a candidate compound to the polypeptide, or interaction of the polypeptide with a substrate in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein (e.g., a glutathione-S-transferase fusion protein) can be provided that adds a domain that allows HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate to be bound to a matrix or other solid support.
In another embodiment, modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell, cell lysate, tissue, tissue lysate, or solution containing a nucleic acid encoding HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is contacted with a candidate compound and the expression of appropriate mRNA or polypeptide (e.g., variant(s)) in the cell, cell lysate, tissue, or tissue lysate, or solution, is determined. The level of expression of appropriate mRNA or polypeptide(s) in the presence of the candidate compound is compared to the level of expression of mRNA or polypeptide(s) in the absence of the candidate compound, or in the presence of the candidate compound vehicle only. The candidate compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the mRNA or polypeptide expression. The level of mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting mRNA or polypeptide.
This invention further pertains to novel compounds identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use a compound identified as described herein in an appropriate animal model. For example, a compound identified as described herein (e.g., a candidate compound that is a modulating compound such as an antisense nucleic acid molecule, a specific antibody, or a polypeptide substrate) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a compound. Alternatively, a compound identified as described herein can be used in an animal model to determine the mechanism of action of such a compound. Furthermore, this invention pertains to uses of novel compounds identified by the above-described screening assays for treatments as described herein. In addition, a compound identified as described herein can be used to alter activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or to alter expression of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), by contacting the polypeptide or the nucleic acid molecule (or contacting a cell comprising the polypeptide or the nucleic acid molecule) with the compound identified as described herein.
Pharmaceutical Compositions
The present invention also pertains to pharmaceutical compositions comprising nucleic acids described herein, particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and/or other variants encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)); and/or comprising a compound that alters (e.g., increases or decreases) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression or HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity as described herein. For instance, a polypeptide, protein, fragment, fusion protein or prodrug thereof, or a nucleotide or nucleic acid construct (vector) comprising a nucleotide of the present invention, a compound that alters HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity, a compound that alters HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid expression, or an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrate or binding partner, can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit the mode of administration.
Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
Methods of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal. Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devises (“gene guns”) and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other compounds.
The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active compound. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., that are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The compound may be incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
Compounds described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The compounds are administered in a therapeutically effective amount. The amount of compounds that will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms of a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, that notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the compounds can be separated, mixed together in any combination, present in a single vial or tablet. Compounds assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each compound and administered in FDA approved dosages in standard time courses.
Methods of Therapy
The present invention also pertains to methods of treatment (prophylactic, diagnostic, and/or therapeutic) for a cell proliferation disease, an apoptotic disease, or a cell differentiation disease, using an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compound. An “HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compound” is a compound that alters (e.g., enhances or inhibits) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity and/or HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule expression, as described herein (e.g., an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) agonist or antagonist). HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compounds can alter HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity or nucleic acid molecule expression by a variety of means, such as, for example, by providing additional HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or by upregulating the transcription or translation of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule; by altering post-translational processing of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide; by altering transcription of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) variants; or by interfering with HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide activity (e.g., by binding to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide), or by downregulating the transcription or translation of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule. Representative HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compounds include the following: nucleic acids or fragments or derivatives thereof described herein, particularly nucleotides encoding the polypeptides described herein and vectors comprising such nucleic acids (e.g., a nucleic acid molecule, cDNA, and/or RNA, such as a nucleic acid encoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or active fragment or derivative thereof, or an oligonucleotide; for example, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, which may optionally comprise at least one polymorphism, or a nucleic acid encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or fragments or derivatives thereof); polypeptides described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 SEQ ID NO: 10 and/or other variants encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or fragments or derivatives thereof); HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) substrates; peptidomimetics; fusion proteins or prodrugs thereof; antibodies (e.g., an antibody to a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an antibody to a non-mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an antibody to a particular variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), as described above); ribozymes; other small molecules; and other compounds that alter (e.g., enhance or inhibit) HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid expression or polypeptide activity, for example, those compounds identified in the screening methods described herein, or that regulate transcription of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) variants (e.g., compounds that affect which variants are expressed, or that affect the amount of each variant that is expressed. More than one HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compound can be used concurrently, if desired.
The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compound that is a nucleic acid is used in the treatment of a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. The term, “treatment” as used herein, refers not only to ameliorating symptoms associated with the disease, but also preventing or delaying the onset of the disease, and also lessening the severity or frequency of symptoms of the disease. The therapy is designed to alter (e.g., inhibit or enhance), replace or supplement activity of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in an individual. For example, an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compound can be administered in order to upregulate or increase the expression or availability of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or of specific variants of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), or, conversely, to downregulate or decrease the expression or availability of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or specific variants of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). Upregulation or increasing expression or availability of a native HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or of a particular variant could interfere with or compensate for the expression or activity of a defective gene or another variant; downregulation or decreasing expression or availability of a native HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule or of a particular variant could minimize the expression or activity of a defective gene or the particular variant and thereby minimize the impact of the defective gene or the particular variant.
The HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compound(s) are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease). The amount that will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
In one embodiment, a nucleic acid of the invention (e.g., a nucleic acid encoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, such as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, which may optionally comprise at least one polymorphism, or a nucleic acid that encodes an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or a variant, derivative or fragment thereof, such as a nucleic acid encoding the protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10) can be used, either alone or in a pharmaceutical composition as described above. For example, HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) or a cDNA encoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, either by itself or included within a vector, can be introduced into cells (either in vitro or in vivo) such that the cells produce native HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. If desired, cells that have been transformed with the gene or cDNA or a vector comprising the gene or cDNA can be introduced (or re-introduced) into an individual affected with the disease. Thus, cells that, in nature, lack native HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression and activity, or have mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression and activity, or have expression of a disease-associated HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) variant, can be engineered to express an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide or an active fragment of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide (or a different variant of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide). In a preferred embodiment, nucleic acid encoding the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide, or an active fragment or derivative thereof, can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal. Other gene transfer systems, including viral and nonviral transfer systems, can be used. Alternatively, nonviral gene transfer methods, such as calcium phosphate coprecipitation, mechanical techniques (e.g., microinjection); membrane fusion-mediated transfer via liposomes; or direct DNA uptake, can also be used to introduce the desired nucleic acid molecule into a cell.
Alternatively, in another embodiment of the invention, a nucleic acid of the invention; a nucleic acid complementary to a nucleic acid of the invention; or a portion of such a nucleic acid (e.g., an oligonucleotide as described below), can be used in “antisense” therapy, in which a nucleic acid (e.g., an oligonucleotide) that specifically hybridizes to the RNA and/or genomic DNA of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) is administered or generated in situ. The antisense nucleic acid that specifically hybridizes to the RNA and/or DNA inhibits expression of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecule, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
An antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above. When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA that encodes an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. Alternatively, the antisense construct can be an oligonucleotide probe which is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and/or genomic DNA of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). In one embodiment, the oligonucleotide probes are modified oligonucleotides that are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, thereby rendering them stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy are also described, for example, by Van der Krol et al., Biotechniques 6: 958-976 (1988); and Stein et al., Cancer Res 48: 2659-2668 (1988). With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g. between the −10 and +10 regions of an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid sequence, are preferred.
To perform antisense therapy, oligonucleotides (RNA, cDNA or DNA) are designed that are complementary to mRNA encoding an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide. The antisense oligonucleotides bind to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required. A sequence “complementary” to a portion of an RNA, as referred to herein, indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
The oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotides can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or compounds facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad Sci. USA 84: 648-652 (1987); PCT International Publication No. WO88/09810)) or the blood-brain barrier (see, e.g., PCT International Publication No. WO89/10134), or hybridization-triggered cleavage agents (see, e.g., Krol et al., BioTechniques 6: 958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent).
The antisense molecules are delivered to cells that express HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in vivo. A number of methods can be used for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically. Alternatively, in a preferred embodiment, a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g., pol III or pol II). The use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) transcripts and thereby prevent translation of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above. For example, a plasmid, cosmid, YAC, or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systematically).
Endogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression can also be reduced by inactivating or “knocking out” HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid sequences or their promoters using targeted homologous recombination (e.g., see Smithies et al., Nature 317: 230-234 (1985); Thomas and Capecchi, Cell 51: 503-512 (1987); Thompson et al., Cell 5: 313-321 (1989)). For example, a mutant, non-functional HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (either the coding regions or regulatory regions of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS)) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS). The recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above. Alternatively, expression of non-mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) can be increased using a similar method: Targeted homologous recombination can be used to insert a DNA construct comprising a non-mutant, functional HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (e.g., a gene having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, which may optionally comprise at least one polymorphism), or a portion thereof, in place of a mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in the cell, as described above. In another embodiment, targeted homologous recombination can be used to insert a DNA construct comprising a nucleic acid that encodes an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide variant that differs from that present in the cell.
Alternatively, endogenous HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) (i.e., the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) promoter and/or enhancers) to form triple helical structures that prevent transcription of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in target cells in the body. (See generally, Helene Anticancer Drug Des., 6(6): 569-84 (1991); Helene et al., Ann, N.Y. Acad. Sci., 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992)). Likewise, the antisense constructs described herein, by antagonizing the normal biological activity of one of the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) proteins, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures. Furthermore, the antisense techniques (e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to an HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA or gene sequence) can be used to investigate role of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in developmental events, as well as the normal cellular function of HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in adult tissue. Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
In yet another embodiment of the invention, other HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) therapeutic compounds as described herein can also be used in the treatment or prevention of a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. The therapeutic compounds can be delivered in a composition, as described above, or by themselves. They can be administered systemically, or can be targeted to a particular tissue. The therapeutic compounds can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade et al.), for example, and can be isolated using standard means such as those described herein.
A combination of any of the above methods of treatment (e.g., administration of non-mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptide in conjunction with antisense therapy targeting mutant HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) mRNA; administration of a first variant encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) in conjunction with antisense therapy targeting a second encoded by HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS), can also be used.
In another embodiment, the invention is directed to HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecules and HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides for use as a medicament in therapy. For example, the nucleic acid molecules or polypeptides of the present invention can be used in the treatment of a cell proliferation disease, an apoptotic disease, or a cell differentiation disease. In addition, the HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) nucleic acid molecules and HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), or HDRP(ΔNLS) polypeptides described herein can be used in the manufacture of a medicament for the treatment of a cell proliferation disease, an apoptotic disease, or a cell differentiation disease.
The invention will be further described by the following non-limiting examples. The teachings of all publications cited herein are incorporated herein by reference in their entirety.
Exemplification
Cloning of cDNA Encodes a Novel HDAC, Designated HDAC9
HDAC9 was cloned by PCR and 3′ rapid amplification of cDNA ends using primers designed from the sequence of human chromosome 7 whose translated product exhibited 80% identity to the HDAC domain of HDAC4, described in detail as follows.
Database analyses indicate that HDRP is located on chromosome 7 (7p15-p21). The human genome database (February 2001 release) of GenBank was searched using the human HDAC4 amino acid sequence. The TBLASTN program was used to identify open reading frames downstream of HDRP on chromosome 7 that exhibit significant homology to the HDAC domain of HDAC4. Several fragments whose translated products exhibit over 58% identity were retrieved. Two sense primers (OL486, 5′-CCATGGAAACGGTACCCAGCAGGC-3′ (SEQ ID NO: 16) and OL487, 5′-CACTCCATCGCTATGATGAAGGG-3′ (SEQ ID NO: 17)) and antisense primers (OL484, 5′-AGTTCCCTTCATCATAGCGATGG-3′ (SEQ ID NO: 18) and OL485, 5′-AATGTACAGGATGCTGGGGT-3′ (SEQ ID NO: 19)) each were designed based upon one of these fragments whose translated products matched amino acids 842-873 of HDAC4. RT-PCR was performed using each of the antisense primers and a sense primer (5′-CCCTTGTAGCTGGTGGAGTTCCCTT-3′ (SEQ ID NO: 20)) from the coding region of HDRP and human brain cDNA as a template. PCR was performed in a Biometra TGRADIENT Thermocycler for 30 cycles at 95° C. for 20 seconds, 60° C. for 20 seconds, and 72° C. for 120 seconds.
3′-rapid amplification of cDNA ends was performed using the sense primer OL486 and adaptor primer 1 (Clontech), and marathon-ready cDNA from human brain (Clontech, Palo Alto, Calif.) according to the manufacturer's instruction. The products were re-amplified using nested sense primer OL487 and adaptor primer 2 (Clontech, Palo Alto, Calif.). PCR products were cloned into pGEM®-T-easy vector (Promega, Madison, Wis.) and sequenced using an automated DNA sequencer at the DNA Sequencing Core Facility of the Memorial Sloan-Kettering Cancer Center, using DNA sequencing methods known to one of skill in the art.
Two cDNAs were cloned from the above-described methods. One cDNA (SEQ ID NO:1) encodes an HDAC9 protein that is 1011 amino acids in length. The other cDNA (SEQ ID NO: 3) encodes an HDAC9a protein that is 879 amino acids long. The cDNA sequence and amino sequence of HDAC9 and HDAC9a are shown in
Each of the HDAC9 and HDAC9a nucleic acid sequences were cloned into the pFLAG-CMV-5b vector (Sigma) in frame with the C-terminal FLAG tag. Only the coding regions plus three extra base pairs (ACC) of cDNA of the HDAC9 and HDAC9a nucleic acid sequences were included in the constructs. These constructs are referred to herein as HDAC9-FLAG and HDAC9a-FLAG, respectively. These constructs are contained in E. coli, and can readily be expressed. For HDAC9, the insert is 3033 bp and for HDAC9a, the insert size is 2637 bp. Both HDAC9 and HDAC9a can be released with EcoRV and BamHI (whose sites have been incorporated in the primers to obtain HDAC9 and HDAC9a coding cDNA for cloning purpose) restriction enzyme digestion.
The HDAC9 cDNA sequences from the known 5′-end of HDRP cDNA to the 3′-untranslated region cloned in this study cover over 511 kb of genomic DNA on chromosome 7. As shown in
Further analysis revealed that exon 7, which contains a nuclear localization signal (NLS) is alternatively spliced in an HDRP isoform, creating HDRP(ΔNLS). RT-PCR analyses using primers based on sequences from exon 6 and exon 14 indicate that this alternative splicing event also occurs in HDAC9 and/or HDAC9a. Thus, it is possible that at least 6 proteins can be generated from a single HDAC9 gene by alternatively splicing of its RNA. The cDNA sequences and amino acid sequences for HDAC9, HDAC9a, HDAC9(ΔNLS), HDAC9a(ΔNLS), and HDRP(ΔNLS) are shown in
HDAC9 mRNA is Differentially Expressed Among Human Tissues
The expression of HDAC9 mRNA was determined by Northern blot analysis using a human multiple tissue Northern blot (Clontech, Palo Alto, Calif.). Hybridization was performed according to the manufacturer's instruction using ExPressHyb solution (Clontech, Palo Alto, Calif.). The 32P-random priming labeled 3′-untranslated region common to both HDAC9 and HDAC9a that shares no significant sequence homology with HDRP was used as a probe. Two transcripts at 9.8 and 4.1 kb were detected in all tissues examined (
The distribution of alternatively spliced mRNA variants among tissues was examined by RT-PCR using primers (OL516 5′-TGTGTCATCGAGCTGGCTTC-3′ (SEQ ID NO: 21) and OL517 5′-ATCTTCTGCAAGTGGCTCCA-3′ (SEQ ID NO: 22)) spanning the alternatively spliced exon 22 and cDNA panel from the same tissues as the multiple tissue Northern blot. PCR was performed in a Biometra TGRADIENT Thermocycler for 30 cycles at 95° C. for 20 seconds, 60° C. for 20 seconds, and 72° C. for 60 seconds. The expected sizes of PCR products were 680 base pairs for HDAC9 and 993 base pairs for HDAC9a. The ratio of HDAC9 and HDAC9a transcripts differed among tissues (
HDAC9 and HDAC9a Possess Histone Deacetylase Activity
HDAC9 was named based on sequence homology to HDAC4 (
C-terminal FLAG-tagged HDAC9 (HDAC9-FLAG) and HDAC9a (HDAC9a-FLAG) expression vectors were constructed using the pFLAG-CMV-5b vector (Sigma) and PCR amplified coding regions of HDAC9 and HDAC9a in frame with the FLAG-tag to form pFLAG-CMV-5b-HDAC9 (plasmid VR1) and pFLAG-CMV-5b-HDAC9a (plasmid VR2). All constructs were confirmed by DNA sequencing.
Transfection of human kidney 293T cells, immunoprecipitation using anti-FLAG M2 Agarose (Sigma), Western blot analyses and dual luciferase assays were performed essentially as previously described by Zhou et al. (Proc. Natl. Acad. Sci. USA, 97:1056-1061 (2000)). Briefly, the cells (American Type Culture Collection) were cultured in DME HG medium (GIBCO/BRL) supplemented with 10% (vol/vol) FBS at 37° C. in a 5% CO2 atmosphere. Transient transfection was performed by using LIPOFECTAMINE® (GIBCO/BRL) or FUGENE® 6 (Roche Molecular Biochemicals) according to the manufacturers' instructions. Cells were harvested 24 to 48 hours after transfection and lysed in IP lysis buffer (50 mM Tris HCl, pH 7.5/120 mM NaCl/5 mM EDTA/0.5% NP-40) at 5×107 cells per ml. Immunoprecipitation with anti-FLAG M2-agarose (Sigma, St. Louis, Mo.) was performed according to the manufacturer's instructions. Immunoprecipitated proteins were released from the agarose beads by using FLAG-peptide and either used directly for HDAC enzymatic activity assays or resolved on SDS/PAGE for Western blot analyses. Anti-FLAG antibody was purchased from Sigma (St. Louis, Mo.). Western blot analyses were performed using standard methods.
HDAC9 and HDAC9a enzymatic activity were assessed with the HDAC Fluorescent Activity Assay/Drug Discovery Kit-AK-500 (BIOMOL Research Laboratories) using a FLUOR DE LYS™ that contains an acetylated lysine side chain as a substrate and immunoprecipitated HDAC9-FLAG and HDAC9a-FLAG polypeptides according to the manufacturer's instruction and a SPECTRAmax® GEMINI XS microplate spectrofluorometer using the SOFTmax® PRO system (Molecular Devices) at excitation 355 nm and emission 460 nm with a cut off filter of 455 nm. Briefly, HDAC9-FLAG and HDAC9a-FLAG were incubated with the substrate overnight at room temperature in a 96-well plate. The reaction was stopped by addition of Fluor De Lys™ Developer and samples were read with the fluorometer.
As shown in
HDAC9 and HDAC9a enzymatic activity was also determined through HDAC enzymatic assays using 3H-histones isolated from murine erythroleukemia cells as a substrate. This assay was performed essentially as described by Richon et al. (Proc. Natl. Acad. Sci. USA, 95:3003-3007 (1998)). Briefly, HDAC9-FLAG and HDAC9a-FLAG were incubated with 3H-histones overnight at 37° C. The reaction was stopped by the addition of 1M HCl/0.1 acetic acid. Released 3H-acetic acid was extracted with ethyl acetate and quantified by scintillation counting. For inhibition studies, the immunoprecipitated complexes were preincubated with the different HDAC inhibitors for 30 minutes at 4° C.
As shown in
HDAC9 and HDAC9a Repress MEF2-Mediated Transcription
The Xenopus homolog of HDRP, MITR, was identified as a MEF2 interacting transcriptional repressor (Sparrow et al., EMBO J. 18:5085-5098(1999)) and mouse HDRP also interacts with and represses MEF2 mediated transcription (Zhang et al., J. Biol. Chem. 276:35-39 (2001)). We first tested whether HDAC9-FLAG and HDAC9a-FLAG interact with MEF2. 293 cells were transfected with vector, HDAC9-FLAG, or HDAC9a-FLAG. The cells were subsequently lysed and HDAC9-FLAG and HDAC9a-FLAG proteins were immunoprecipitated with anti-FLAG antibodies. Western blot analysis of the immunoprecipitated proteins was carried out, using anti-MEF-2 antibody to probe the blot. As shown in
It was then determined whether HDAC9 and HDAC9a repress MEF2-mediated transcription. This determination was carried out as follows. The p3XMEF2-luciferase reporter gene (100 ng) and the vector pRL-TK (Promega) (5 ng) were co-transfected into 293T cells in the absence (pcDNA3 empty vector) or presence of MEF2C (100 ng of pCMV-MEF2C). HDAC9-F (1 ng, 10 ng, or 100 ng of pFLAG-HDAC9; pFLAG-HDAC9 and HDAC9-FLAG are different constructs, with the FLAG sequence located at opposite ends of the HDAC9 nucleotide, but are functionally equivalent) or HDAC9a-F (1 ng, 10 ng, or 100 ng of pFLAG-HDAC9a; pFLAG-HDAC9a and HDAC9a-FLAG are different constructs, with the FLAG sequence located at opposite ends of the HDAC9a nucleotide, but are functionally equivalent) was included in a subset of experimental groups with the MEF2C vector. pFLAG empty vector was used to adjust the DNA to an equal amount in each transfection. The cells were harvested 24 to 36 hours after transfection and the luciferase activities were measured using the Dual-Luciferase™ Reporter Assay System from Promega according to the manufacturer's instruction. The firefly luciferase activity was first normalized to the co-transfected Renilla luciferase activity (encoded by the pRL-TK vector), and the luciferase activity value for cells transfected with MEF2C alone was set at 1. MEF2C activated transcription over 30 times the basal level of transcription. As shown in
Described herein is the identification and characterization of a new class II HDAC, designated HDAC9. HDAC9 has several alternatively spliced isoforms, one of which is the previously identified HDRP (Zhou et al., Proc. Natl. Acad. Sci. USA 97:1056-1061 (2000)). HDAC9 and HDAC9a possess HDAC activity, which appears to have a lower specific enzymatic activity than HDAC4. While not wishing to be bound by any particular theory, it is possible that an essential co-factor is lost during immunoprecipitation or does not exist in 293T cells (for example, metastasis-associated protein 2 is essential for the assembly of a catalytically active HDAC1 (Zhang et al., Genes Dev. 13:1924-1935 (1999)), the substrates used are not its natural substrate, or the FLAG tag which interferes with the folding of the protein.
Searching the human genome with the HDAC domain from either HDAC1 or HDAC9 identified a total of 10 HDACs in the presently completed human genome sequence, a number of which are schematically represented in
All references described herein are incorporated by reference in their entirety. While this invention has been particularly shown and described with reference to preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Richon, Victoria M., Marks, Paul A., Rifkind, Richard A., Zhou, Xianbo
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| Aug 13 2002 | RICHON, VICTORIA M | Sloan-Kettering Institute for Cancer Research | CHANGE OF ADDRESS OF ASSIGNEE | 024841 | /0578 | |
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| Aug 15 2002 | MARKS, PAUL A | Sloan-Kettering Institute for Cancer Research | CHANGE OF ADDRESS OF ASSIGNEE | 024841 | /0578 | |
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| Sep 10 2002 | ZHOU, XIANBO | Sloan-Kettering Institute for Cancer Research | CHANGE OF ADDRESS OF ASSIGNEE | 024841 | /0578 | |
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